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The effects of high pressure on thermolysin activity and spectroscopic properties were studied. Thermolysin distinct pressure-induced activation with a maximum observed at 200-250 MPa for a dipeptide amide substrate and at 100-120 Mpa for a heptapeptide substrate. By examining the pressure dependence of the hydrolic rate for the former substrate using a high pressure stopped-flow apparatus as a mixing device under elevated pressures, the activation volume of the reaction was -71 ml mol-1 at 25 °C. ΔV was accompanied by a negative activation expansibility and a value of -95 ml mol-1 was obtained at 45°C. A prolonged incubation of thermolysin under high pressure, however, caused a time-dependent deactivation. These changes due to pressure were monitored by several spectroscopic methods. The fourth-derivative absorbance spectrum showed an irreversible change, mostly in the tyrosine and tryptophan regions, at a pressure higher than 300 Mpa. Intrnsic fluoresnce and circular dichrosim measurements of thermolysin in solution also detected irreversible changes. All these measurements indicated that a change occurred at higher pressures and are explained by a simple two-state transition model accompanied by a large, negative change in the volume of reaction. [ABSTRACT FROM AUTHOR]

Basic fibroblast growth factor (bFGF), a potent mitogen for arterial smooth muscle cells has been shown to play a fundamental role in the pathogenesis of arteriosclerosis and restenisos by stimulating the proliferation of vascular smooth muscle cells. We found that partially phosphorothioate-modified 15-redisue antisense oligodeoxynucleotides complementary to bFGF mRNA at 0.1-2.0 μM clock growth and division of cultured human and bovine coronary smooth muscle cells I a dose-dependent manner. The effect is sequence specific at low (0.1-0.5 μM) nontoxic concentrations. It is associated with inhibition of expression of pericellular and intracellular bFGF, with a decreased de novo synthesis of bFGF and is partly reversible by the addition of exogenous (recombinant) bFGF. The antisense effect lasts 48-72h and diminishes therafter. If the antisense oligodeoxynucleotide medium is replaced by an oligonucleotide-free medium after 24 h. the [³H]thymidine incorporation rate returns to control levels. Under the same conditions, the corresponding sense oligodeoxynucleotide exerts negligible nonspecific inhibitoty actions. The antiproliferative potency of the 15-residue antisense oligodeoxynucleotide is markedly enhanced by adding 3-4 nobase-pairing guanosine residues at the 5'- and 3'-termini of the 15-residue antisense oligonucleotide. [ABSTRACT FROM AUTHOR]

The (2'-5')oligoandenylate synthetae represents a family of interferon-induced proteins which when activated by double-stranded (ds)RNA polymerized ATP into 2'-5'-linked oligomers with the general formula pppA(2'p5A)n, where n>1, which for convenience are referred to as 2-5A. We studied here the influence of pH. DsRNA concenration and time on oligomeric compositio of 2-5A synthesized by purified 69-kDa and 100-kDa isoforms of (2'-5')oligo(adenylate) synthetase. In optimal conditions form activity, the 69-kDa form synthesized higher oligomers of 2-5A molecules whereas the 100 kDa form synthesized preferentially dimeric molecules, which are known not to be functional for the activation of Rnase L. This difference does not reflect a differential affinity of the enzymes for the preformed 2-5A dimmer, which is found to be a very poor substrate for both enzymes. This latter strongly suggests that the mechanism of elngation is more likely processive. Moreover, we show that both isoforms have efficient nucleotidyl-transferase activity and provide evidence that, in optimized conditions, GTP can be used alone as substrate by these enzymes to generate pppG2'p5'G. Our results clearly demonstrate that the 69-kDa and 100-kDa forms of )2'-5')oligoadenylate synthase manifest various differential catalytic activities, and favor the hypothesis that these enzymes might have other functions in the cell besides those in the 2-5A system. [ABSTRACT FROM AUTHOR]

The fadH gene coding for an NADPH-dependent 2,4-dienoyl-CoA reductase from Escherichia coli has been cloned by the polymerase chain reaction. This gene is located at 67.65 min on the E. coli chromosome. The complete open reading frame contains 2019 bp coding for the processed protein of 671 amino acid residues, with a calculated molecular mass of 72.55 kDa, which lacks the N-terminal methionine. Construction and expression of the plasmid pNDH, which contained the fadH gene under the control of the T7 promoter, resulted in a 110-fold increase in the reductase activity above the level detected in E. coli cells containing the control vector. The kinetic parameters of the purified reductase were determined to be 50 µM and 2.3 µM for the Km values of NADPH and 2-trans, 4-trans-decadienoyl-CoA, respectively, and 16 s-1 for the km value. Analysis of the kinetic data revealed that the reaction catalyzed by this enzyme proceeds via a ping-pong mechanism. The observed dissimilarity between the E. coli and mammalian 2.4-dienoyl-CoA reductase sequences suggests that they have evolved from distinct ancestral genes. Sequence analysis also suggests that the N-terminal part of the E. coli reductase contains the FAD-binding domain whereas the NADPH-binding domain is located in the C-terminal region of the protein. [ABSTRACT FROM AUTHOR]

Analytical dark-field scanning transmission electron microscopy (STEM) of freeze-dried unstained specimens of keyhole limpet henocyanin (KLH: from Megathura crenulata. A prosobranch gastropod) gave a molecular mass of kDa for the subunit of KLH1 and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/Page. Within the 400-kDa KLH1 subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45-60 kDa functional domains (termed 1a through 1h) and determined their sequential arrangement. The KLH1 domains differ biochemically and immunologically from each other and from the previously characterized seven domains of KLH2 (termed 2a through 2g). Our partial amino acid sequences suggest that a domain, equivalent to the C-terminal domain 1h, is missing in KLH2. This deficiency is believed to be genuine and not an artifact of the subunit preparation procedure, since STEM measurements of the native didecamers yielded a mass difference of about 800 kDa between KLH1 and KLH2 (8.3 Mda versus 7.5 Mda), correlating with 20 copies of a financial copies to a functional 1h domain. It was also shown that the KLH1 didecamer can be rapidly split (minutes) into a almost homogeneous population of stable decamers by increasing the pH of the Tris/saline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl2 and 5mM MgCl2, to pH 8.5. reformation of the didecamers occurred more slowly (days) upon analysis against the pH7.4 stabilizing buffer leads to the more rapid (overnight) formation of didecamers together with a significant number of previously observed KLH1 multidecamers, which could be structurally distinguished from the established multidecamers of KLH2. [ABSTRACT FROM AUTHOR]

Lipopolysacharides (LPS) from antigentically different strains assigned to seroroups O:3 and O:6 of Helicobacter pylori were isolated as water-soluble material of high Mr and as water-insoluble gels of low Mr. Chemical and spectroscopic analyses of the soluble LPS and oligosaccharides liberated from the water-insoluble gels led to proposed structures with Lewis (Le) antigen determinants terminating regular repeating units of different types, linked in turn to inner core regions of invariable structure. The O:6 LPS has two populations of related molecules with chains of 3-linked D-glycero-α-D-manno-heptose residues similar to those in the MO19 strain, one with and the other without a single terminal Lewis (Ley) epitope. In contrast, in the O:3 LPS. Lewis (Lex and Ley epitopes terminate a partially fucosylated N-acetyllactosaminoglycan, but a heptan chain similar to that in the O:6 LS was shown to connect the outer chains to the inner core. These PS provide examples of the molecular mimicry of cell-surface glycoconjugates. Structural variations of LPS between strains, and differences in some aspects of structure within the strains, between high Mr and low Mr LPS indicate a class of LPS whose mechanisms of biosynthesis lead to overall architectures different from those characteristics of most LPS from enteric bacteria. [ABSTRACT FROM AUTHOR]

Very-low density lipoprotein receptor (VLDLR) belong to the low-density lipoprotein receptor gamilty of endocytosis receptors. It binds a variety of different ligands, including apolipoprotein E, Mr-40000 receptor-associated-protein (RAP), and some serine proteinase/erpin complexes. We previously demonstrated the occurrence of two forms of VLDLR in SDS/PAGE, migrating with M.r 105000 and M.r 130000, respectively [Heegaard. C. W., Simonsen, A.C.W., Oka. K., Kjøller. L., Christensen. A., Madsen. B. Ellgaard. L., Chan. L. & Andreasen, P.A. (1995) J. Biol. Chem. 270. 20855-20869] We know demonstrate that these two forms correspond to forms with the absence (type-II) and presence (type-I) of the O-linked glycosylation domain encoded by exon 16, respectively. We show that the two forms have the same binding affinity to RAP and serine proteinase/serpin complexes. Using reverse transcription and PCR, we demonstrate that the splice variation giving rise to the two forms is highly cell specific. In particular, we demonstrate that human breast carcinomas-express predominantly or exclusively the variant lacking exon 16. By immunohistochemistry, we demonstrte that VLDLR is mainly expressed by the epithelial cancer cells in these carcinomas. The VLDLR variant expressed by epithelial cancer cells could function in the clearance of cell-associated serine proteinase/serpin complexes in breast carcinomas. [ABSTRACT FROM AUTHOR]

Bacilllus subtilis Ffh protein is a homologue of the 54-kDa subunit of mammalian signal recognition particle (SRP54). It contains three highly hydrophobic regions (h1, h2, and h3) in the C-terminal methionine domain (M-domain). Two of the dydrophobic regions, h2 and h3, are essential for small cytoplasmic RNA (scRNA) binding [Kurita, K., Honda, K., Suzuma. S., Takamatsu, H., Nakamura K., & Yamane. K. (1996) J. Biol. Chem. 271, 13140-13146]. Using purified presecretory proteins and mutant Ffh proteins, we identified a region required for presecretory protein binding in B. subtilis Ffh. Deletion of this region, which consisted of residues Ser311-Gly362 of B. subtilis Ffh, including a hydrophobic sequence (h1), reduced precursor binding activity. In contrast, deletions of residues Leu121-Lys279, Lys364-Met446, or Leu338-Ser397 of B. subtilis Ffh did not. We also analyzed the mutant B. subtilis Ffh protens, FfhQQOr and Ffh QQQQ having wild-type residues 398-401 (Arg-Arg-Lys-Arg) replaced with Gln3Arg and Gln4, respectively. FfhQQOR bound to both scRNA and presecretory protein. Although the FfhQQQQ mutation prevented binding to scRNA, binding to the precursor was not affected. FfhQQOR restored the growth of B. subtilis DF46 strain in which ffh gene expression is regulated by an inducible promoter in the absence of an inducer, whereas FfhQQQQ did not. These results indicate that the region including h1 is required for B. subtilis Ffh to bind to presecretory protein. The results also suggests that scRNA is required for the complete function of the B. subtilis SRP-like particle in vivo, although this protein is intrinsically capable of binding a signal peptide free from scRNA. [ABSTRACT FROM AUTHOR]

A human monocyte-activating CC chemokine has been identified based on sequences in an expressed sequence tag (EST) cDNA database. The protein shows highest sequence identity to th macrophage inflammatory protein (MIP) group of chemokines, particularly MIP-3 (76.7$) and MIP-1α (75.4%), and has been named MIP-5. Model building confirms that the protein has a similar three dimensional structure to other chemokines, but has an additional third disulphide bond. Northern blot analysis and reverse-transcriptase PCR show that the mRNA for MIP-5 is expressed at a high levels in liver, intestine and in lung leukocytes. MIP-5 induces chemotaxis of human monocytes. T-lymphocytes and, to a lesser degree, eosinophilsat nanomolar concentrations; it has no effect on neutophil migration. In receptor-binding assays. MIP-5 shows IC50 values of 12 mM for competition with 125I-MIP-1α for binding to CC-chemokine receptor (CCR)1, and 2.5 nM for competition with 125I-MCP-3 for inding to CCR3. It shows no ability to compete with ligand for binding to the two interleukin (IL)-8 receptors (CXC-chemokine receptors 1 and 2) or to CCR2, CCR4 or CCR5. Consistent with this binding data, MIP-5 wa sonly able to induce calcium fluxes in CHO cells stably transfected with CCR1 or CCR3. [ABSTRACT FROM AUTHOR]

Phosphorylation of decorin was investigated by incubating a rat fibroblast cell line with radiolabelled phosphate and carbohydrate precursors. There was a transient phosphorylation of the linkage-region saccharides in intracellular decorin prior to assembly of the galactosaminoglycan chain. Phosphorylation gradually increased from xylosylated, galactosyl-xylosylated to galactosyl-galactosyl-xylosylated core protein where all triasaccharide stubs were phosphorylated. Addition of the first glucuronate residue was accompanied by rapid dephosphorylation. Brefeldin A treatment resulted in segregation of galactosaminoglycan synthesis and dephosphorylation chain [Moses, J., Oldberg,Å., Eklund, E. & Frasson, L.-A. (1997) Eur. J. Biochem., in the press] by using chondroitin AC lyase and chondro-glycuronidase, followed by β-galactosidase treatment, demonstrated the sequence galactosyl-phosphoxylose. The xylose was resistant to direct periodate oxidation, but sensitive after treatment with alkaline phosphatase, showing that the phosphate was located at C2 of xylose. The transient 2-phosphorylation of xylose may be involved in intracellular transport and/or in the control of modifications of the glycan chain. [ABSTRACT FROM AUTHOR]

Sulfite stimulates the rate of ATP hydrolysis by the ATP synthse in chromatophores of Rhodobacter capsulatus. The stimulated activity is inhibited by oligomycin. The activation takes place also in incoupled chromatophores. The activation consists in an increase of about 12 - 15-fold of the Vmax for the ATP hydrolysis reaction, while the Km for MgATP is unaffected at 0.16 ± 0.03 mM. The dependence of Vmax on the sulfite concentration follows a hyperbolic pattern with half maximum effect at 12mM. Sulfite effects the ability if the enzyme in translocating protons. Concomitant measurements of the rate of ATP hydrolysis and of ATP-induced protonic flows demonstrate that at sulfite concentrations of greater than 10mM the hydrolytic reaction becomes progressively uncoupled from the process of proton translocation. This is accompanied by an inhibition of ATP synthesis, either driven by lights or by artificially induced ionic gradients. ATP synthesis is totally inhibited at concentration of at least 80 mM. Sulfite interferes with the mechanism of activation by Δμ̃H+. Low concentrations of this anion (≤ 2 mM) prevent the activation by Δμ̃H+. At higher concentrations a marked stimulation of the activity prevails, regardless of the occurrence of a Δμ̃H+ across the membrane. Phosphate at millimolar concentrations can reverse the inhibition by sulfite. These experimental results can be stimulated by a model assuming multiple and competitive equilibria for phosphate or sulfite binding with two binding sites for the two ligands (for sulfite K1S = 0.26 and K2S, = 37 mM, and for phosphate K1P = 0.06 and K2P = 4.22 mM), and in which the state bound only to one sulfite molecule is totally inactive in hydrolysis. The competition between phosphate and sulfite is consistent with the molecular structures of the two ligands and of the enzyme. [ABSTRACT FROM AUTHOR]

Gangliosides, constituents of surfaces if vertebrate cells, modulate important cellular functions. Ganglioside-specific sialidases that possibly control these prosesses have been observed in a number of tissues, but their characterization has proved difficult due to their low abundance and lability. Here we describe the partial isolation and characterization of a ganglioside sialidase from human brain grey matter. After membrane extraction with octyglucoside, the enzyme ws purified about 1300-fold by ion-exchange, affinity and gel-permeation chromatographies. Although PAGE still showed several protein bands, specific photoaffinity labeling with iodinated 5-N-acetyl-9-(4-azidosalicolyamido)-2,9-dideoxy-2, 3-didehydroneuraminic acid identified a single polypeptide of 60 kDa likely to contain the active site of the sialidase. In the presence of 0.4% octyglucoside, the purified sialidase desialylated gangliosides GM3, GDia>, GT1bb>, but was inactive towards GM1, GM2 colominic acid, sialyl-(α2-3)-lactose, 2-(4-methylumbelliferyl)-neuraminate, or the glycoprotein fetuin. The ganglioside sialidase activity was strongly inhibited by 2-deoxy-2.3-didehydro-N-acetylneuminic acid, heparin and heparan sulfate. Because of tis substrate ad inhibitor profiles, the purified resembles the activity characterized previously in the plasma membrane of human neuroblastoma cells, but is distinct from a lysosomal activity. The purified brain sialidase thus appears to function in the selective desialylation of gangliosides with terminal sialic acid residues. [ABSTRACT FROM AUTHOR]

The kinetics of triplex formation between the oligonucleotides d(AGGGAGG-GAGGAAGGGAGGG) (20AG), D(TGGGTGGGTGGTTGGGTGGG) 20GT) and a 29-bp polypurine-polypyrimidine sequence located in the c-Ki-ras promoter (D) was studied by electrophoretic experiments in 50 mM Tris/acetate, pH 7.4.50 mM NaCl, 5 mM MgCl2. Rates of triplex formation were determined at three different termperatures (20°C, 37 °C and 45 °C), under pseudo-first order conditions obtained by using the triplex-forming oligonucleotide (TFO) 500-fold in excess over the target duplex (5 nM). Measurements at TFO/target ratios of 20 and 100 were also carried out. At 37 °C the pseudo first-order constants, kabc were 18.9x10-5-1 for 20AG and 13.0x10-5 s-1 for 20GT, yielding association half-lives of 1 h and 1.5 h, respectively. Second-order association constants were found to be n the order of 10²M-1s-1; these are slightly lower if compared with those measured for triplex formation by polypyrimidine (C,T) oligonucleotides (10³M-1s-1 [Maher. L. J., Dervan. P.B. & Wolf. B.J. (1990) Biochemistry 29, 8820-8826: Xodo. L. E. (1995) Eur. J. Biochem. 228, 918-926; Bates. P. J., Dosanjh. H. S., Jenkins. T. C., Laughton. C. A. & Neidle. S. (1995) Nucleic Acids Res. 23, 3627-3632] but dramatically lower when compared with duplex recombination from complementary stands (106 M-1 s-1 [Craig. M. E. & Doty. P. (1971) J. Mol. Biol. 62, 383-401: Pörschke. D. & Eigen. M. (1971) J. Mol. Biol. 62, 361-381. Dissociation rate constants k-1, were indirectly obtained from equilibrium contants (kd and found to be, at 37° 6.7x10-7 s-1 and 5.4x10-6 s-1 for 20AG and 20GT, respectively. From the rate constants obtained at, 20°C, 37 °C and 45 °C we estimated activation energies of triplex formation between D plus 20AG and Dplus 20GT of respectively 134 ± 29 and 88 ±21 kJ/mol. Moreover, the activation energies for the reaction of triplex dissociation were 385 ±50± kJ/molfor 20AG and 330 ± 42kJ/mol for 20GT. Decreasing the TFO/target ratio from 500 abd 100 or 20, we observed a concomitant decrease of the association rate, in keeping with the finding that triplex formation occurs through a bimolecular process. We found that the effect of salt on triplex formation is rather complex, as, the addition of 2 mM spermidine boosted the binding rate of 20GT, but slightly reduced that 20AG; the increase of NCl from 50 mM to 100 mM or 150 mM decreased the rate of triplex formation. Finally, the biological implications of the kinetic behaviour exhibited by the two triplex formation. Finally, the biological implications of the kinetic behaviour exhibited by the two triplex-forming oligonucleotides specific for the c-Ki-ras promoter are discussed. [ABSTRACT FROM AUTHOR]

Upon hererologous expression of the Brevibacterium ammoniagenes type-1 fatty acid synthase FAS-A in Escherichia coli. Only the pantetheine-free apoenzyme is synthesized. Activation of FAS-A to its holoform was achieved by transformation with a second B. ammoniagene, PPT1. encoding a type-1 FAS-specific phosphopanthetheine transferase. PPT1 was identified as a coding sequence located immediately downstream of the second FAS gene present on the B. ammoniagenes genome, fasB. Due to this linkage. PPT1 was part of the cloned fasB DNA region and, consequently, FAS-B but not FAS-A was synthesized as holoFAS in E. coli. PPT1 encodes a protein of 153 amino acids and has a calculated molecular mass of 16884 Da. The PPT1 gene product contains 25% identical and 42% conserved amino acids compared with the type-II acyl-carries-protein-activating enzyme of E. coli. Although there is essentially no intergenic region between fasB and PPT1, the PPTase gene is autonomously expressed in E. coli if flanked by 200 bp of its endogenous. 5' DNA. The structural independence of Ppt1p was confirmed immunologically, as specific antibodies react with the purified PPTase but not with FAS-B. Overexpression and purification of the His-tagged Ppt1p allowed the in vitro activation of apoFAS-A. This holoenzyme synthesis requires, in addition to Ppt1p. CoA and Mg2+ and leads to a specific FAS activity comparable to that of natural B. ammoniagenes FAS-A. The reactivity of the in vitro-activated FAS-A was verified by the optical FAS assay and by analysis of its in vitro products. In agreement with the known overall colinearity of B. ammoniagenes FAS-B and the Saccharomytes cerevisiae FAS1 and FAS2 gene products, a PPT1-like sequence is also observed at the C terminus of FAS2. However, in contrast to B.ammoniagenes PPT1, this sequence is a integral part of the yeast FAS2 gene. Thus, activatio of type-1 fatty acid synthases may be accomplished by distinct tans-acting PPTase enzymes and by intrinsic cis-acting PPTase domains. [ABSTRACT FROM AUTHOR]

The hfb2 gene encoding the hydrophobin HFBII of the filametous fungus Trichiderma reesei was isolated by heterologoous hybridization using the vegetative hydrophobin I, hfb1, gene of T. reesei as a probe. The hfb2 gene codes for a typical fungal secretd hydrophobin of 71 amino acids conataining eight cysteine residues. The amino acid similarity towards HFBI is 69%. The HFBII protein was isolated from the fungal spores by extraction with trifluoroacetic acid/acetonitrile solution, and by bubbling from the lactose-based culture medium. Expression of the hfb1 and hfb2 genes is divergent, hfb1 expression was only observed in vegetative cultures on glucose-containing and sorbitol-containing media. It was not expressed on media containing complex plant pilysaccharides, cellulose, xylan, cellobiose or lactose, whereas hfb2 was highly expressed in vegetative cultures on these media. Expression of hfb2 was also strongly induced by N and C starvation, by light and in conidiating cultures. [ABSTRACT FROM AUTHOR]

Random mutagenesis with ouabain selection used to comprehensively scan the extracellular and transmembrane domains of the α1 subunit of the sheep Na+/K+-ATPase for amino acid residues that alter ouabain sensitivity. The four random mutant libraries used in this study include all of the transmembrane and extracellular regions of the molecule as well as 75% of the cytoplasmic domains. Through an extensive number of HeLa cell tranfections of these libraries and subsequent ouabain selection, 24 ouaain-resistant clones have been identified. All previously described amino acids that confer ouabain resistance were identified, confirming the completeness of this random mutagenesis screen. The amino acid substitutions that confer the greatest ouabain resistance, such as Gln111→Arg, Asp121→Gly, Asp121→Glu, Asn122→Asp, and Thr797→Ala were identified more than once in this study. This extensive survey of the extracellular and transmembrane regions of the Na+/K+-ATPase molecule has identified two new regions of the molecule that effect ouabain sensitivity: the H4 and the H10 transmembrane regions. The new substitutions identified in this study are Leu330→Gln. Ala331→gly. Thr338→Ala, and Thr338→Asn in te H4 transmembrane domain and Phe982→Ser in the H10 transmembrane domain. These substitutions confer modest increases in the concentration of cardiac glycoside needed to produce 50% inhibition of activity (IC50values), 3.1 - 7.9-fold difference. The results of this extensive screening of the Na+/K+-ATPase α1 subunit to identify amino acids residues that are important in ouabain sensitivity further supports our hypothesis that the H1-H2 and H4-H8 regions represent the major binding sites for the cardiac glycoside class of drugs. [ABSTRACT FROM AUTHOR]

The non-specific serine-protease inhibitor protein-C inhibitor (PCI) inactivates its target enzymes by forming stable 1:1 complexes. Heparin stimulates most PCI/protease reactions, but interferes with the inhibition of tissue kallikrein by PCI by a hitherto unknown mechanism. In this study we analyzed the inhibitory effect of heparin on the tissue-kallikrein - PCI interaction. Free PCI an tissue-kallikrein · PCI complexes but not free tissue kallikrein bound to heparin-Sepharose, implying that the inhibitory effect of heparin cannot be caused by a tissue-kallikrein - heparin interaction. Hepain did not dissociate tissue-kallikrein · PCI complexes, making it unlikely that in the presence of heparin PCI becomes a substrate for, rather than inhibitor of, tissue kallikrein. However, heparin-bound PCI, which was able to form complexes with 125I-urokinse, did not form complexes with 125I-tissue-kallikrein. This suggests that the inhibitory effect of heparin is either based on the neutralization of positive charges in the PCI molecule, which might be required for the interaction of PCI with the acidic protease tissue kallikrein, or on a change in reactivity of PCI upon heparin binding, making heparin-bound PCI no longer a tissue-kallikrein inhibitor. Neutralization of basic amino acids in the PCI molecule by glumatic acid, which prevented in a dose-dependent way the inhibitory effect of heparin, did not have any effect on the tissue-kallikrein &mdashl PCI interaction. Therefore, direct involvement of bsic amino acid residues present in the heparin-binding site of PCI in the tissue-kallikrein — PCI interaction can be excluded. Heparin binding might rather cause a change in reactivity of PCI (e.g. by inducing a conformational change or by steric interference), thereby preventing its interaction with tissue kallikrein. [ABSTRACT FROM AUTHOR]

We studied the relationship between sphingomyelin, calcium, spermine and sphingosine in regulation of phospholipase C (PLC)δ1 activity. Inhibition of PLC δ1 by sphingomyelin was promoted by spermine and Ca2+ and was partially abolished by sphingosine. The effect of sphingosine and spermine entirely depended on Ca2+. In the absence of Ca2+, no effect of these substances on PLC δ1 activity was observed. Using deletion mutants and active fragments of PLC δ1 generated by limited proteolysis , we have studied the structural requirements of the enzyme for regulation by these compounds. The deletion mutant of PLC δ1 lacking the first 58 amino acids and the mutant lacking the entire pleckstrin homology (PH) domain were fully active in the detergent assay, and their activities were affected by spermine, sphingosine, Ca2+ and sphingomyelin to the same extent as the native enzyme. The limited proteolysis of PLC δ1 generated two fragments of 40 kDa and30 kDa, which formed a stable active complex. The relationship between Ca2+ concentration and enzyme activity was almost identical for the native PLC δ1 and the proteolytic complex. The activity of the proteolytic complex formed by the kDa and 30 kDa peptides was not affected by spermine and sphingosine. Sphingomyelin inhibited the complex slightly less than the native PLC δ1, and this inhibition was not promoted by spermine. These observations suggest that for activation of PLC δ1 by spermine and sphingosine, the region spanning domains of high conservation, named X and Y, must be intact. In contrast, the PH domain and the intact spanning region of the X and Y domains are not essential for inhibition of PLC δ1 by sphingomyelin. [ABSTRACT FROM AUTHOR]

Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativa L.) which was assumed m initiate storage-globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa. whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25 244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2 % identical to sulfhydrylendopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna mungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of 1-day-germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A. the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of the unusual diheme split-Soret cytochorome c from the sulphate-reducing Desulfovibrio desulfuricans strain ATCC 27774 has been determined using classical chemical sequencing techniques and mass spectrometry. The 247-residue sequence shows almost no similarity with any other known diheme cytochrome c. but the heme-binding site of the protein is similar to that of the cytochromes c3 from the sulphate reducers. The cytochrome-c-like domain of the protein covers only the C-terminal part of the molecule, and there is evidence for at least one more domain containing four cysteine residues, which might bind another cofactor, possibly a non-heme iron-containing cluster. This domain is similar to a sequence fragment of the genome of Archaeoglobus fulgidus, which confirm the high conservation of the genes involved in sulfate reduction. [ABSTRACT FROM AUTHOR]

The product of the nusB gene of Escherichia coli modulates the efficiency of transcription termination at nut (N utilization) sites of various bacterial and bacteriophage γ genes. Similar control mechanisms operate in eukaryotic viruses (e.g. human immunodeficiency virus). A recombinant strain of E. colt producing relatively large amounts of NusB protein (about 10% of cell protein) was constructed. The protein could be purified with high yield by anion-exchange chromatography followed by gel-permeation chromatography, The protein is a monomer of 15.6 kDa as shown by analytical ultracentrifugation. Structural studies were performed using protein samples labelled with 15N. 13C and ²H in various combinations. Heteronuclear three-dimensional triple-resonance NMR experiments combined with a semi-automatic assignment procedure yielded the sequential assignment of the [H. 13C and 15N backbone resonances. Based on experimentally derived scalar couplings, chemical-shift values, amide-exchange data, and a semiquantitative interpretation of NOE data, the secondary structure of NusB bas classified as α helical, comprising seven α helices. [ABSTRACT FROM AUTHOR]

The yeast tmsl gene was originally identified as a multi-copy suppressor of a lethal growth arrest caused by expression of a tumour mutant cDNA of p53 in fission yeast. The tmsl gene product (Tms1) was found to form stable complexes with p53 in yeast and in vitro; using purified recombinant proteins., the interaction was mapped the C-terminal region of p53 from a latent to an activated state capable of transactivating various cellular genes involved in growth suppression or apoptosis. Since there is evidence for an evolutionary conservation of a Tms-1related protein in mammals, the effect of a phosphorylation status of the C-terminus of p53 on Tms1/p53 complex formation in vitro has been investigated. Whereas mutants changing the edc2 phosphorylation site at position 315 of human p53 had only little effect on Tms1/p53 complex formation, we found that mutants involving the protein kinase CK2 site at position 392 showed a significantly decreased relative affinity for the Tms1 protein. The same result was obtained by using a C-terminal fragment of p53 which was phosphorylated by purified protein kinase CK2, suggesting that the complex formation of p53 with cellular C-terminal binding proteins like Tms1 impairs regulation by phosphorylation. [ABSTRACT FROM AUTHOR]

Site-directed mutagenesis was used to assess the contribution of individual residues and a bound calcium in the 55 - 69 region of the thermolysin-like protease of Bacillus stearothermophilus (TLP -ste) to thermal stability. The importance of the 55 - 69 region was reflected by finding the almost all mutation had drastic effects on stability. These effects (both stabilizing and destabilizing) were obtained by mutation affecting main chain flexibility, as well as by mutations affecting the interaction between the 55 - 69 region and the rest of the protease molecule. The calcium-dependency of stability could be largely abolished by mutating one of its ligands (Asp57 or Asp59). In the case of the Asp57→Ser mutation, the accompanying loss in stability was modest compared with the effects of others destabilizing mutations or the effects of (combinations of) stabilizing mutations. The detailed knowledge of the stability-determining region of TLP-ste permits effective rational design of stabilizing mutations, which, presumably, are also useful for related TLP such as thermolysin. This is demonstrated by the successful design of stabilizing salt bridge involving residues 65 and 11. [ABSTRACT FROM AUTHOR]

In the present study, we synthesized 14C-labelled 5-nitro-l.2.4-triazol-3-one (NTO) and investigated its hepatic metabolism by dexamethasone-induced murine hepatic microsomes. Under the nitrogen atmosphere. 5-amino-l.2.4-triazol-3-one was the only detected metabolite of NTO. The microsomal nitroreductase activity was dependent on NADPH, totally inhibited by carbon monoxide and partially inhibited by oxygen. In aerobic conditions, beside a low amount of amine, the major metabolite formed is the 5-hydroxy-triazolone, urazole. This compound resulted from the oxidative denitrification of NTO, which produced equivalent amount of nitrite. This reaction, like the nitroreductase activity, was dependent on NADPH and totally inhibited by carbon monoxide. Both nitroreduction and oxidative denitrification were inhibited by imidazole-related inhibitors: miconazole and methimazole, and to a less extent by N-octylamine. The microsomal denitrification was induced by the treatment of mrs with dexamethasone and phenobarbital. The microsomal reductase activity is present in untreated rat microsomes, and recovered with various inducers. The results of this study indicate the role played by cytochrome P-450 in the metabolism of NTO. supported by its transformation with reconstituted cytochrome P-450 systems. [ABSTRACT FROM AUTHOR]

Phospholipase D (PLD) activity in human embryonic kidney (HEK) cells is stimulated by phorbolester-activated protein kinase C (PKC) and by membrane receptors, the latter apparently cating via the GTP-binding proteins. ADP-ribosylation factor (ARF) and Rho. In the present study, performed in cell-free preparations, we have characterized and compared the regulation of HEK cell PLD activity by the stable GTP analogue, guanosine 5'-O-[γ-thio]triphosphate (GTP[S]), and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In digitonin-permeabilized HEK cells, prelabeled with [³H]oleic acid.GTP[S] and PMA caused an approximately threefold concentration-dependent increase in the formation of [³H]phosphatidylethanol, measured in the presence of ethanol. Neomycin, which is known to complex with the PLD activities with similar sensitivity. GDP and its analogue, guanosine 5'-O-[β-thio]diphosphate, inhibited the stimulatory effect of GTP[S], whereas the PMA response was prevented by the non-selective PKC inhibitor, staurosporine, but not vice versa. PLD stimulation by GTP[S], but not by PMA, was markedly reduced upon cylosol depletion and reconstituted by purified recombinant ARF1. In HEK cell membranes, addition of purified recombinant ARNO, a guanine-nucleotide-exchange factor for ARF1, potentiated the GTP[S]-stimulated PLD activity. PLD stimulation by PMA in HEK cell membranes required MgATP and was largely prevented by the selective PKC inhibitors Goe 6976 abd bisindolylmaletimide I. Immunoblot analysis demonstrated that both conventional PKC (&alpja;, β, γ) and atypical PKC isozymes (&Zigma;, τ) were present in HEK cell membranes. The results indicate that phorbol ester stimulation of PLD activity in HEK cells apparently occurs by a phosphorylation-dependent mechanism involving membrane-associated PKC isozymes but not ARF proteins, the major targets of GTP[S] action. [ABSTRACT FROM AUTHOR]

D-Threonine aldolase is an enzyme that catalyzes the cleavage of D-threonine into glycine and acetaldehyde: Its activity was found in several genera of bacteria such as Arthrobacter. Alcaligenes. Xanthomonas, and Pseudomonas, but not in yeasts or fungi. The enzyme was purified to homogeneity from one strain, Arthrobacter sp. DK-38. The enzyme appeared to consist of a single polypeptide chain with an apparent molecular mass of 51 kDa. This enzyme, as well as L-threonine aldolase, requires pyridoxal 5'phosphate (pyridoxal-P) as a coenzyme. Unlike other pyridoxaI-P enzymes, D-threonine aldolase also requires a divalent cation such as Co2+, Ni2+, Mn2+, or Mg2+ for its catalytic activity. The enzyme completely lost its activity in the absence of either pyridoxal-P or a divalent cation. A divalent cation was also essential for the thermal stability of the enzyme. The metal-free enzyme tends to become thermally unstable, resulting in the inreversible loss of its catalytic activity. The enzyme is strictly D-specific for the α-position, whereas it cannot distinguish between threo and erythro forms at the β-position. Thus, D-threonine and D-allothreonine act as substrates of the enzyme, but their kinetic parameters are different: the Km and Vmax values are 3.81 mM and 38.8 μmol · min-1 · mg-1 toward D-threonine.. and 14.0 mM and 102 μmol · min-1 · mg-1 toward D-allothreonine, respectively. The aldolase reaction is reversible, and the enzyme is therefore able to produce nearly equimolar amounts of D-threonine and D-allothreonine through C-C bond formation between glycine and acetaldehyde. The enzyme also acts, in the same manner, on several other D-β-hydroxy-α-amino acids, including D-β-phenylserine. D-β-hydroxy-α-aminovaleric acid. D-β-3,4-dihydroxyphenylserine. and D-β-3,4-methylenedioxyphenylserine. [ABSTRACT FROM AUTHOR]

An examination of the roots of Lycium chinense (Solanaceae) has resulted in the discovery of 14 catystegines, a cycloheptane bearing an amino group and three hydroxyl groups, and two polyhydroxylated piperidine alkaloids. Calystegines A7, and B5, in addition m the previously known calystegines A3, A5 A6 B1, B2, B3, B4, C1, C2, and N1 ,were isolated and determined as lα, 2β, .4α-trihydroxy-nortropane and lα,2α,4α,7α.-tetrahydroxy-nortropane, respectively, L. chinense also had two polyhydroxytropanes bearing a methyl group on the nitrogen atom, unlike the previously reported nortropane alkaloids. They were established as N-methylcalystegines B2 and C1, and their N-methyl groups were found to be axially oriented from NOE experiments, lβ-Amino-3β,4β 5α-trihydroxycycloheptane was also present in L. chinense and may be a biosynthetic precursor of the calystegines that occur in this plant. Two polyhydroxypiperidine alkaloids, fagomine and 6-deoxyfagomine. were isolated. Calystegine B2, is a potent competitive inhibitor of almond β-glucosidase (K1 = 1.9 μM) and coffee bean α.-gatactosidase (K1 = 0.86 μM), while N-methylcalystegine B2 was a more potent competitive inhibitor of the latter enzyme {K1= 0.47 μM) than the parent compound but showed a marked lack of inhibitory, activities towards most other glycosidases. Since this compound is a very specific inhibitor of α.-galactosidase and inhibits rat liver Iysosomal α.-galactosidase with a K1 of 1.8 μM, it may provide a useful experimental model for the lysosomal storage disorder, Fabry's disease. The addition of a hydroxyl group at C6exo, as in calystegines B1 and C1, enhances the inhibitory potential towards β-glucosidase and β-galactosidase but markedly lowers or abolishes inhibition towards α-galactosidase. Hence, the N-methylation of calystegine C1 did not enhance its inhibition of α-galactosidase. The chemical N-methylation of calystegines A1 and B4 markedly enhanced inhibition of coffee bean α-gatactosidase, with K1 values of 5.2 μM and 36 μM, respectively, but almost eliminated their inhibitory potential towards β-glucosidase and trehalase, respectiveiy. Thus, methylation of the nitrogen atom significantly altered the specificity of the inhibitors. [ABSTRACT FROM AUTHOR]

The effect of sphinosylphosphorylcholine (SphPCho) on the intracellular pH (pH1) in GH4 C1 cells was investigated. SphPCho evoked a very slow increase in basal pH1. In cells acidified with nigericin. SphPCho induced a rapid alkalinization of the cells. The effect was inhibited in a Na+-free buffer solution. but was insensitive to ethylisopropyl amiloride, a potent inhibitor of Na+-H+ exchangers (NHE). Reverse transcription and PCR showed that the predominant isoform of the antiport expressed in GH4C1 cells is NHE-1. The rate of alkalinization after stimulation with propionate, and after addition of Na+ to cells acidified with NH4 C1, was enhanced in cells treated with SphPCho. The initial rate of alkalinization after addition of Na+ to acidified cells treated with SphPCho gave an apparent Km value of 15 ±2 mM for Na+. The Vmax value was 9±2 mM H+/min. The effect was insensitive to ouabain. staurosporine and bafilomycin A. However, the SphPCho-evoked alkalinization was abolished in cells treated with 2-deoxyD-glucose. The effect was not due to the charge of the molecule, as stearylamine increased pH1 in Na+containing and Na+-free buffer. The results show that SphPCho may activate Na+-H+ exchange, and that this effect is mediated via an amiloride-insensitive exchange mechanism. [ABSTRACT FROM AUTHOR]

4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum catalyzes the dehydration of 4hydroxybutyryl-CoA m crotonyl-CoA. Although dehydration is an overall non-redox reaction, the enzyme contains FAD and Fe-S clusters. Previous work has shown that the Fe-S clusters are difficult to reduce and therefore unlikely m be redox-active in catalysis. Here, Mössbauer spectroscopy has been used to characterise the Fe-S clusters in active as well as in air-inactivated enzyme. In zero magnetic field at 80 K and 4.2 K, the spectra of active dehydratase consisted mainly of one species (95 %) with quadrupole splitting, ΔEQ = l.00 mm s-1 and isomer shit δ = 0.43 mm s-1. Magnetically perturbed Mössbauer spectra indicated a spin of zero. In the presence of 6 mM crotonyl-CoA, the spectra remained unchanged. Taken together, the data show that there are [4Fe-4S]2+ in the enzyme, most probably two clusters/ homotetramer. that the four iron atoms in each cluster are coordinated in an identical fashion, and that there is no direct interaction with substrates. We therefore infer that the Fe-S clusters serve a structural rather than a catalytic role in 4-hydroxybutyryI-CoA dehydratase. In air-inactivated enzyme (10% residual activity). a new doublet appeared (58%) with ΔEQ = 0.72 mm ss-1, δ = 0.32 mm s-1 and S = 0. The assignment of this subspectrum to [3Fe-4S]4 clusters, based on the typical Mössbauer parameters, is contradicted by the finding of spin zero for the species. One possible explanation could be spin-coupling of two [3Fe-4S]+ clusters in close proximity. [ABSTRACT FROM AUTHOR]

The fatty acid specificity of phospholipase D purified from germinating sunflower seeds was studied using mixed micelles with variable detergent/phospholipid ratios. The main advantage of this approach is that since the substrate is integrated in the detergent micelles, comparisons can be made between the kinetic constants of a wide range of phosphatidylcholine (PtdCho) compounds with various fatty acid contents. Phospholipase D is subject to interracial activation as it is most active on water-insoluble substrates. It is not active on sphingomyelin and only slightly on lysophosphatidylcholine. By fitting the curves based on the experimental kinetic data, the interfacial dissociation constant of phospholipase D. the maximum hydrolysis rate Vm and the kinetic constant Kbm were determined with the micellar substrate. The specificity of various substrates was examined by comparing the Vm/KKbm values, and it was noted that sunflower phospholipase D is most active on medium-chain fatty PtdCho compounds. With long-chain natural phospholipids, the specificity of phospholipase D was slightly dependent on the level of fatty acid unsaturation. The pure enzyme was able to hydrolyse the sunflower phospholipids present m mixed detergent micelles but not the phospholipids integrated in the natural sunflower oil body structure. We concluded, however, that during the germination of sunflower seeds, phospholipase D might be involved in the degradation of oil bodies, since other factors present in crude seed extracts may make phospholipids accessible to the enzyme. [ABSTRACT FROM AUTHOR]

Human progelatinase B was activated by collagenase-3 in a time-dependent fashion. Activation proceeded through an intermediate form of M, 86000 to the final active form of M, 82000. N-terminal amino acid sequence determination demonstrated that the Glu40-Met41 peptide bond was initially hydrotysed followed by cleavage of the Arg87-Phe88 peptide bond releasing the rest of the propeptide domain which was accompanied by the achievement of maximal enzymatic activity as revealed using a quenched fluorescent substrate. Kinetic analysis of activation revealed that the rates were dependent on the concentration of the proenzyme as well as active collagenase-3. Active gelatinase B did not contribute to the activation rate of the proenzyme initiated by collagenase-3 and our results indicate that progelatinase B activation proceeds via bimolecular cleavage with collagenase-3 involving sequential cleavage of the propeptide in two steps. The activation rates were not dependent on C-terminal domain interactions between progelatinase B and collagenase-3, as assessed using wild-type and C-terminal deletion mutants of both enzymes. Since elevated levels of both gelatinase B and collagenase-3 have been observed in arthritis and breast cancer pathology these enzymes may well form a proteolytic cascade in these diseases which allows rapid turnover of the extracellular matrix. [ABSTRACT FROM AUTHOR]

A homology-modelled structure of dihydrolipoamide dehydrogenase from the halophilic archaeon. Hatoferax volcanii, has been generated using the crystal structure of the enzyme from Pseudomonas fluroscents. Analysis of the halophilic enzyme structure identified a potential K -binding site comprising four co-ordinated glutamate residues (E423 and E426 from each monomer) at the subunit interface of the dimeric protein. Whilst E426 is conserved throughout non-halophilic dihydrolipoamide dehydrogenases. E423 is only present in the halophilic enzyme. Four site-directed mutations of the Haloferax dihydrolipoamide dehydrogenase have been made (E423D, E423Q, E423S, and E423A) and the recombinam mutants expressed and characterised. From an analysis of their kinetic properties, salt-dependent activities and thermal stabilities, it is concluded that this site has art important influence on the halophilicity of the enzyme. The findings support the view that the arrangement and interaction of the negatively changed amino acids are as important as the total net charge in determining the adaptation of proteins to high salt concentrations. [ABSTRACT FROM AUTHOR]

The active H-cluster of the Fe-hydrogenases from Megasphaera elsdenii and Desulfovibrio vulgaris (strain Hildenborough) has been investigated with one- and two-dimensional pulsed EPR spectroscopy. In both complexes the coordination of a nitrogen-containing ligand was found. The unusual quadrupole interaction parameters (D, vulgaris: quadrupole coupling constant. K = 1.20 MHz. asymmetry parameter n = 0.32. M. etsdenii: K = 1.23 MHz, n = 0.25) indicate a non-protein type of nitrogen and are consistent with cyanide as ligand to the H-cluster. The additional interactions measured on the EPR signal of the inactivated H-cluster in D. vulgaris hydrogenase are consistent with an imidazole interaction similar to that found in Rieske-type iron-sulfur clusters. Since a His residue near the putative H-cluster binding motif of Cys residues. His371, is the only conserved His in Fe-hydrogenases. it is a likely candidate for the base that accepts the proton in the heterolytic cleavage of molecular hydrogen. The reactivation of the enzyme is accompanied by direct binding of the imidazole ring to the H-cluster. [ABSTRACT FROM AUTHOR]

The interaction of recombinant ascorbate peroxidase (APX) with its physiological substrate, ascorbate. has been studied by electronic and NMR spectroscopies, and by phenylhydrazine-modification experiments. The binding interaction for the cyanide-bound derivative (APX-CN) is consistent with a 1:1 stoichiometry and is characterised by an equilibrium dissociation binding constant. Kd, of 11.6.&plusmnl;0,4 μM (pH 7.002, μ = 0.10 M, 25.0°C). Individual distances between the non-exchangeable substrate protons of APX-CN and the haem iron were determined by paramagnetic-relaxation NMR measurements, and the data indicate that the ascorbate binds 0.90-1.12 nm from the haem iron. The reaction of ferric APX with the suicide substrate phenylhydrazine yields predominantly (60%) a covalent haem adduct which is modified at the C20 carbon, indicating that substrate binding and oxidation is close to the exposed C20 position of the haem. as observed for other classical peroxidases. Molecular-modelling studies, using the NNMderived distance restraints in conjunction with the crystal structure of the enzyme [Patterson. W. R, & Poulos. T L, (1995) Biochemistry 34, 4331-4341], are consistent with binding of the substrate close to the C20 position and a possible functional role for alanine 134 (proline in other class-III peroxidases) is implicated. [ABSTRACT FROM AUTHOR]

Two new multiheme cytochromes were isolated from the anaerobic sulfur reducing bacterium Desulfuromonas acetoxidans. They have monomeric molecular masses of 50 and 65 kDa and contain six and eight hemes, respectively. Visible and EPR spectroscopies, in the as-isolated (oxidised) cytochromes, show the presence of only low-spin hemes in the 50-kDa cytochrome, and of high-spin and low-spin hemes in the 65-kDa cytochrome. The EPR spectra of the native 65-kDa cytochrome indicate multiple heme—heme interactions, including integer-spin systems as judged by parallel-mode EPR. The 50-kDa cytochrome has a complex redox pattern, as shown by EPR redox titrations, and contains one heine with unusual characteristics. Both cytochromes cover an extremely wide range of reduction potentials, which go from + 100 mV to - 375 mV for the 50-kDa cytochrome, and +185 mV to -235 mV for the 65-kDa cytochrome. The two cytochromes were tested for hydroxylamine oxidoreductase activity and polysulfide reductase activity, but neither displayed any activity. In contrast, it was found for the first time that the previously characterised cytochrome c551.5, from the same bacterium is very active in the reduction of polysulfide, which suggests that it acts as a terminal reductase in D. acetoxidans. [ABSTRACT FROM AUTHOR]

The pattern of phosphorylation of adjacent serine residues in several peptides based on the N-terminal region of human cardiac troponin I has been analysed by PAGE and H NMR spectroscopy to identify the products. With cAMP-dependent protein kinase, Ser24 is rapidly phosphorylated, and subsequent much slower phosphorylation of Ser23 occurs only after phosphorylation of Ser24 is almost complete. Monophosphorylation of the peptide al Ser23 was not detected at any time. On replacement of Arg22 with Ala or Met the sole phosphorylation target was Ser23, phosphorylation being considerably slower than for Ser24 in the wild-type peptide, while diphosphorylation could not be detected after prolonged incubation. The results emphasise the importance of the N-terminal sequence RRRSS for the function of cardiac troponin I and imply that in human cardiac muscle unstimulated by adrenaline, troponin 1 is phosphorylated on Ser24. Comparative two-dimensional NOESY data indicate that in the diphosphorytared form at physiological pH values, specific structural constraints are imposed on the N-terminal peptide region. These constraints result in the effective screening of the two phosphate groups from each other by the arginine residues N-terminal to the serine pair and stabilisation of the structure in the region of residues 25-29, which is adjacent to a site of interaction between troponin I and troponin C. These conformational changes presumably underlie the decrease in calcium sensitivity of the myofibrillar ATPase that occurs after adrenaline intervention. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus I (HIV-1) protease is a prime target in the search for drugs to combat the AIDS virus. The enzyme functions as a C2-symmetric dimer, cleaving the gag and gag-pol viral polyproteins at distinct sites. The possession of a twofold axis passing through the active site, has led to the design of C2-symmetrical inhibitors in the form of substrate-based transition-state analogs. One of the most active compounds of this class of inhibitors is HOE/BAY 793, which contains a vicinal diol central unit [Budt, K.-H., Hansen, J., Knolle, J., Meichsner, C., Paessens, A., Ruppert, D. & Stowasser, B. & Winkler, I. (1990) European Patent application EP0428.849; Budt. K.-H., Hansen, J., Knolle, I., Meichsner, C., Ruppert, D., Paessens, A. & Stowasser B. (1993) [Xth International Conference on AIDS; Budt, K.-H., Peyman. A., Hansen. J., Knolle, J., Meichsner. C., Paessens. A., Ruppert. D. & Stowassen B. (1995) Bioorg. Med Chem. 3, 559-571.] The structure of this inhibitor bound to HIV-1 protease, in two different crystal forms, has been solved at 0.24-nm resolution using X-ray crystallography. In both forms, the details of the inhibitor-protease interactions revealed an overall asymmetric binding mode, especially between the central diol unit and the active-site aspartates. The main binding interactions comprise several specific H-bonds and hydrophobic contacts, which rationalize many of the characteristics of the structure/ activity relationship in the class of vicinal diol inhibitors. In a general analysis of the mobility of the flap regions, which cover the active site and participate directly in binding, using our structures and the HIV protease models present in the Brookhaven databank, we found that in most structures the flexibility of the flaps is limited by local crystal contacts. However, in one of the structures presented here, no significant crystal contacts to the flap regions were present, and as a result the flexibility of the inhibitor bound flaps increased significantly. This suggests that the mobility and conformational flexibility of the flap residues are important in the functioning of HIV-1 protease, and must be considered in the future design of drugs against HIV protease anti in structure-based drug design in general. [ABSTRACT FROM AUTHOR]

Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (α and/or α') and two non-catalytic, β-subunits. The β-subunit possesses antagonist functions that can be physically dissected by generating synthetic fragments encompassing its N-terminal and C-terminal domains. Here we show that by mutating basic residues in the 74-77 and in the 191- 198 regions of the α-subunit, the negative regulation by the β-subunit and by its N-terminal synthetic fragment CK2β-(41-77), which is observable using calmodulin as a substrate for phosphorylation, is drastically reduced. In contrast, the positive regulation by a C-terminal CK2β-(155-215)-peptide is unaffected or even increased. Moreover, the basal activity of α mutants K74-77A, K79R80K83A. and R191R195K198A toward specific peptide substrates is stimulated by the β-subunit many fold more than rival of α wild type, while extrastimulation by β mutant D55L56E57A, observable with α wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of β is mediated by basic residues in the 74-83 and in the 191-198, sequences of the α-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the β subunit operates as a pseudosubstrate. In contrast, another CK2α mutant, V66A, is more sensitive to inhibition by either β-subunit or its N-terminal. CK2β(1 - 77)-peptide, while its stimulation by the C-terminal peptide, C K 2β-(155- 2l5 ), is comparable to that of a wild type. These observations suggest an indirect rote of Val66, in conferring to the α-subunit a conformation less sensitive to down regulation by β-subunit. [ABSTRACT FROM AUTHOR]

Very-low-density lipoprotein receptor (VLDLR) and α2-macroglobulin receptor/low-density-lipoprorein-receptor-related protein (α2MR/LRP) are multifunctional endocytosis receptors of the low-density lipoprotein receptor family. Both have been shown to mediate endocytosis and degradation of complex between plasminogen activators and type-1 plasminogen-activator inhibitor (PAI-1) by cultured cells. We have now studied the specificity of binding and endocytosis by VLDLR and α2MR/LRP among a variety of serine proteinase/serpin complexes, including various combinations of the serine proteinases urokinasetype and tissue-type plasminogen activators, plasmin, thrombin, human leukocyte elastase, cathepsin G, and plasma kallikrein with the serpins PAI-l, horse leukocyte elastase inhibitor, protein C inhibitor, C1inhibiton α2-antiplasmin, α1-proteinase inhibitor, α1-antichymotrypsin, protease nexin-1, heparin cofactor II, and antithrorabin III, Binding was estimated with radiolabelled ligands in ligand blotting analysis and microtiter well assays. Endocytosis was estimated by measuring receptor-associated protein (RAP)-sensitive degradation of radiolabelled complexes by Chinese hamster ovary cells transfected with VLDLR cDNA and by COS-1 cells, which have a high endogenous expression of α2MR/LRP. We found thai the receptors bind with high affinity to some, but not all, combinations of plasminogen activators and thrumbin with PAI-1, protease nexin-1, protein C inhibitor, and antithrombin III, while complexes of many serine proteinases with their primary inhibitor, i.e. plasmin/α2-antiplasmin complex, do not bind, or bind with a very low affinity. Both the serine proteinase and the serpin moieties contribute to the binding specificity. The binding specificities of VLDLR and α2MR/LRP are overlapping, but not identical. The results suggest that VLDLR and α2MR/LRP have different biological functions by having different binding specificities as well as by being expressed by different cell types. [ABSTRACT FROM AUTHOR]

Prokaryotic mycothiol-dependent formaldehyde dehydrogenase has been structurally characterized by peptide analysis of the 360-residue protein chain and by molecular modelling and functional correlation with the conformational properties of zinc-containing alcohol dehydrogenases. The structure is found to be a divergent medium-chain dehydrogenase/reductase (MDR), at a phytogenetic position intermediate between the cluster of dimeric alcohol dehydrogenases of all classes (including the human forms), and several tetrameric reductases/dehydrogenases. Molecular modelling and functionally important residues suggest a fold of the mycothiol-dependent formaldehyde dehydrogenase related overall to that of MDR alcohol dehydrogenases, with the presence of the catalytic and structural zinc atoms, but otherwise much altered active-site relationships compatible with the different substrate specificity, and an altered loop structure compatible with differences in the quaternary structure. Residues typical of glutathione binding in class-III alcohol dehydrogenase are not present, consistent with that the mycothiol factor is not closely similar to glutathione. The molecular architecture is different from that of the 'constant' alcohol dehydrogenases (of class-Ill type) and the 'variable' alcohol dehydrogenases (of class-I and class-II types), further supporting the unique structure of mycothiol-dependent formaldehyde dehydrogenase. Borders of internal chain-length differences between this and other MDR enzymes coincide in different combinations, supporting the concept of limited changes in loop regions within this whole family of proteins. [ABSTRACT FROM AUTHOR]

Purine-purine mismatches can base-pair in a variety of configurations depending on solution conditions. The G · G mismatch, which also occurs in the G-quartet structure, has been shown by both x-ray crystallography and NMR to adopt G(anti) · G(syn) mispairs, with very different hydrogen bonding patterns [Skelly, J., Edwards, K., Jenkins, T. C. & Neidle, S. (1993) Proc. Natl Acad. Sci. USA 90, 804–808; Cognet, J. A. H., Gabarro-Arpa, J., Le Bret, M., van der Marel, G. A. van Boom, J. H. & Fazakerley, G. V. (1991) Nucleic Acids. Res. 19, 6771–6779] while we have recently suggested the presence of weakly hydrogen-bonded G(anti) · G(anti) pairs in solution [Borden, K. L. B., Jenkins, T. C., Skelly, J. V., Brown, T. & Lane, A. N. (1992) Biochemistry 31, 5411–5422]. Spectral overlap and additional exchange processes have made detailed structural analysis difficult in these mismatched oligomers. We have used NMR to characterise the conformations of four duplexes containing single G · G mismatches, including a nonamer d(CATCGGATG), two undecamers d(GCATTGAATGC) and d(CATGTGACGTG) that can each form a self-complementary duplex with a single G · G mispair in the centre, and the non- self-complementary d(GTAACGACATG) · d(CATGTGGTTAC). The three self complementary duplexes have a single set of NMR resonances, and all four duplexes show evidence of conformational exchange at the mismatch site. The N1H resonances of the mismatched G residues each integrate to two protons, ruling out the enol tantomer. They resonate between 10.5 10.7 ppm, far upfield of the Watson-Crick hydrogen-bonded GN1H and exchange readily with water protons. Intraresidue GH8-H1′ NOE intensities are two-threefold larger tot the mismatched G residues than in G · C base pairs, indicating the presence of syn conformations. NOE time courses for the self-comple- mentary duplexes were consistent with an equimolar mixture of G(syn) · G(anti) and G (anti) · G(syn) states. By symmetry, these states must be interconverting at a rate that is fast on the chemical shift timescale. In the non-self-complementary undecamer, the NOE data indicated that the distinguishable mismatched G residues also spend a significant, but different, fraction of the time in both the syn and anti conformations. The rate constant for the syn/anti transition in the non-self-complementary undecamer was determined as ≈ 14000 s-1 at 303 K from rotating frame T1 measurements, and the apparent frequency difference was > 250 Hz. Calculations based on NOEs and coupling constants showed that the duplexes are overall in the B form. Improved agreement with the NOE data for the mismatched residues could be obtained by constructing linear averages of conformations for the mismatched bases. [ABSTRACT FROM AUTHOR]

The C-terminus of α-spectrins contains two putative calcium-binding sites or EF-hands. To characterize the binding, we have isolated clones from a human fetal liver cDNA library and expressed several fragments comprising either one or both of these sites. When the isolated clones were sequenced, we found that three consecutive nucleotides differed compared to the published sequence. The discrepancy affected two codons in the first of the two putative calcium sites. These codons translated into glutamate and phenylalanine, which are identical to the residues present at the same position in other α-spectrins. In the presence of magnesium, only recombinant peptides comprising the second putative site bound calcium as determined by a calcium overlay assay. Although the first putative EF-hand appeared to bind some calcium in the absence of magnesium, no binding could be detected under stringent conditions. Therefore. it is likely that the second EF-hand constitutes the only functional calcium-binding site in the C-terminus of human non-erythroid α-spectrin. Since peptides comprising the second EF-hand bound calcium nearly as well as intact spectrin, it is also apparent that the second EF hand constitutes the major binding site for calcium in spectrin. The relative change in negative ellipticity, induced by the binding of calcium, indicates a dissociation constant of approximately 120 μM. [ABSTRACT FROM AUTHOR]

A comparative study of the reactivity of histidine residues towards diethyl pyrocarbonate performed on iron-free human and rabbit serotransferrins, human and bovine lactotransferrins and chicken ovotransferrin revealed important differences in the kinetic data and in the accessibility of the histidine residues involved in each of the two iron-binding sites. At a low concentration of reagent (2 mM), the reaction of serotransferrins and lactotransferrins was biphasic and of pseudo first-order. The rate constants for the fast reacting histidine residues were k1 > 0.9 min-1 for the lactotransferrins and only 0.25 min-1 for serotransferrins and ovotransferrin. The fast reactivity of the histidines of lactotransferrins allows the specific destruction of only one of the iron-binding sites using a low diethyl pyrocarbonate concentration (less than 6 mM). The same result was obtained with serotransferrins using a higher reagent concentration (10-12 mM). The behavior of ovotransferrin was quite different since the total iron-binding capacity was lost in presence of 10 mM diethyl pyrocarbonate. The increase of the molarity of diethyl pyrocarbonate up to 25 mM failed to modify all the histidine residues and induced an N,N'-dicarbethoxylation as well as a O-carbethoxylation of tyrosine residues. The use of differential molar absorptivities for the quantification of modified residues has led to the demonstration of the involvement of only one histidine residue in the accessible binding site of human and bovine lactotransferrins, and at least two residues in the accessible binding site of human and rabbit serotransferrins. The inability to modify the histidine residues of the second iron-binding site of serotransferrins and lactotransferrins may be due to a buried localization of this site. [ABSTRACT FROM AUTHOR]

Lycopene cyclization to γ-carotene and then to βcarotene is partially blocked in Phycomyces by the presence of carR mutations in heterokaryosis or by the addition of 2-(4-chlorophenyl)thiotriethylamine MCI to the medium. We have quantitatively determined the carotenes synthesized by Phycomyces under conditions of partially blocked cyclization and in the presence of either carα mutations in heterokaryosis or polyoxyethylenesorbitan monooleate (Tween-SO) in the medium. We conclude that transfer of intermediate substrates between the enzyme aggregates involved in carotenogenesis does occur in the wild type but not in heterokaryons for car&Aalpha; mutations, and is facilitated by Tween-80. [ABSTRACT FROM AUTHOR]

A protein phosphatase was isolated from the yeast, Candida utilities, which could reactivate (dephosphorylate) the phosphorylated form of the NAD-dependent glutamate dehvdrogenase. The protein could also dephosphory1ate casein, histone and kemptide (a heptapeptide corresponding to the phosphorylation site of liver pyruvate kinase).Reactivation of the phosphorylated glutamate dehydrogenase was stimulated by the simultaneous addition of NAD and L-glutamate; 2-oxoglutarate, NH4+ and NADH had no effect. The reactivation of phosphorylated glutamate dehydrogenase could be inhibited by phosphate, pyrophosphate and fluoride. [ABSTRACT FROM AUTHOR]

30-S ribosomal subunits of Escherichia coli form a stable complex with MS2 RNA or Qβ RNA at 37βC in the absence of initiator tRNA. The complex functions as a precursor of initiation since it can enter the ribosome cycle in the presence of inhibitors of de novo initiation. [ABSTRACT FROM AUTHOR]

1. The consequences of incubating liposomes with rat thymocytes have been studied using liposomes of dipalmitoylphosphatidylcholine and cholesterol or dipalmitoylphosphatidylcholine only. 2. Dipalmitoylphosphatidylcholine-cholesterol liposomes do not bind to the cells and can be removed by washing. An increase in cellular cholesterol is observed. However dipalmitoylphosphatidylcholine liposomes bind rapidly to the cells and cannot be removed by repeated washing. Cholesterol is removed from the cells. 3. There are small changes in intracellular cations in the cholesterol-enriched cells, but no transport studies have been made. Cells depleted of cholesterol lose K+ with little change in intracellular Na+. Na+ influx is increased. The majority of this increase appears to be ouabain-sensitive, indicating an increase in pump-mediated Na+ influx. K+ influx is reduced. 4. The significance of these results is discussed. [ABSTRACT FROM AUTHOR]

The deoxyoctanucleotide (5′-3′)d(A-A-G-G-A-G-G-T), which is complementary to the 3′ end of 16-S RNA, inhibits the formation of the complex between the 30-S subunit and MS2 RNA described in the preceding paper. If the complex is preformed, the octanucleotide cannot prevent entry of the complex into the ribosome cycle upon supplementation with the components for protein synthesis. The subunit · MS2-RNA complex is unable to bind the octanucleotide. It is concluded that in the subunit · phage-RNA initiation precursor the 16-S terminus is base-paired with a complementary MS2 RNA sequence. Edeine, aurintricarboxylic acid and antibodies against ribosomal protein S1 prevent the association of phage RNA with 30-S subunits. These compounds do not, however, inhibit the binding of (5′-3′)d(A-A-G-G-A-G-G-T) to 30-S subunits. It is concluded that the formation of the complex between MS2 RNA and 30-S subunits does not depend solely on the Shine and Dalgarno base-pairing reaction. [ABSTRACT FROM AUTHOR]

The subunit structure of membrane-associated spectrin was investigated by crosslinking human erythrocyte membranes with a variety of cleavable photosensitive heterobifunctional reagents by flash photolysis millisecond crosslinking. Crosslinked complexes were analyzed on agarose-polyacrylamide gels whose high exclusion limit permits the resolution of molecules with Mr larger than 106. Crosslinking of membrane ghosts produced a series of new bands with apparent molecular weights of approximately 420000, 710000 and 910000, as well as a band at the top of the gels. No significant bands were detectable between the 910000-Mr band and the material at the gel top. The molecular weights of the three bands correspond to spectrin dimer, trimer, and tetramer, suggesting a limiting tetrameric stoichiometry for membrane-associated spectrin. The rate of formation of spectrin oligomers was examined by increasing crosslinking time. Dimers accumulate before trimer and tetramer, and as tetramer increased, dimer diminished. A simple kinetic model is employed which is also consistent with spectrin tetramer existing on the cytoplasmic surface of the erythrocyte membrane as the basic structural unit. [ABSTRACT FROM AUTHOR]

1. The interaction of chicken ovoinhibitor with pancreatic trypsin, chymotrypsin and elastase I was studied in the absence of substrate by affinity chromatography, electrophoresis and gel filtration. 2. It was found that ovoinhibitor has, at least, five separate and non-overlapping binding sites: two for trypsin, two for chymotrypsin and one for elastase. Most likely, all five sites may be occupied simultaneously. 3. Electrophoretic studies indicated that two binding sites for trypsin and probably also for chymotrypsin differ in their affinity toward the respective enzyme. [ABSTRACT FROM AUTHOR]